1. Field of the Invention
This invention relates to two novel pentadecatrienyl resorcinol hydroxylases which are in the cytochrome P450 gene superfamily. This invention relates to the DNA sequences of these novel enzymes, the amino acid sequences of these novel enzymes, expression vectors containing the DNA sequences of these novel enzymes, methods of making these novel enzymes, methods of transforming plants to express these novel enzymes, and transgenic plants expressing these novel enzymes which may result in the production of dihydrosorgoleone which may be secreted by a plant and undergo oxidation to become sorgoleone.
2. Prior Art Description
Allelopathy, sometimes referred to as a form of chemical warfare between plants, can be defined as the production and release of chemical substances by one species that inhibit the growth of another species (Inderjit and Duke, Planta 217:529-539 (2003); Weston and Duke, Crit. Rev. Plant Sci. 22:367-389 (2003)). Allelopathic interactions have been proposed to have profound effects on the evolution of plant communities through the loss of susceptible species via chemical interference, and by imposing selective pressure favoring individuals resistant to inhibition from a given allelochemical (e.g., Schulz and Wieland, Chemoecology 9:133-141 (1999)). Furthermore, allelopathic compounds released by grain crop species are thought to play a significant role in cover crops or within intercropping systems where they act as weed suppressants. Allelopathic compounds have been characterized in a number of plants such as black walnut, wheat, rice, and sorghum (Bertin, et al., Plant and Soil 256: 67-83 (2003); Inderjit and Duke, (2003); Duke et al., Outlooks Pest Management 16: 64-68 (2005)). These chemicals which can be produced and released by many types of plants, algae, bacteria, and fungi and which often involve secondary metabolites, are referred to as allelochemicals or phytotoxins when produced by plants. Of note, allelopathy and allelochemicals can also positively influence the growth, survival, and reproduction of other organisms.
Despite the ecological and agronomic importance of allelochemicals, relatively few pathways have been characterized in detail at the molecular level. One notable exception is the identification and characterization of all the genes encoding the enzymes responsible for the biosynthesis of 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one, a benzoxazinoid, in Zea mays (Frey et al., Science 277:696-699 (1997)). Benzoxazinoids are thought to act as alleopathic chemicals in the rhizosphere, in addition to being defense compounds against microbial pathogens and insect herbivores (Sicker et al., Int. Rev. Cytol. 198:319-346 (2000); Friebe, J. Crop Prod. 4:379-400 (2001)).
Several Sorghum species have been reported to produce phytotoxins (secondary metabolites) which are exuded from their root systems into the rhizosphere, which suppress the growth of competing species (Einhellig, Agronomy Journal 88:886-893 (1996)). Numerous studies have contributed to the discovery and identification of the chemicals that are responsible for this observed allelopathic inhibition. For example, studies on the biologically-active components of both water-soluble and water-insoluble exudates from roots of Sorghum bicolor have demonstrated their role in the growth inhibition of lettuce seedlings (Lactuca sativa), as well as a number of important invasive weed species (Netzly and Butler, Crop Science 26:775-778 (1986)). The major constituent of these exudates was identified as 2-hydroxy-5-methoxy-3-[(Z,Z)-8′,11′,14′-pentadecatriene]-p-benzoquinone, referred to as sorgoleone (Chang, et al., J. Am. Chem. Soc. 108:7858-7860 (1986)), which has been estimated to account for as much as 85% of the exudate material (w/w) in some varieties of sorghum (Czarnota, et al., Weed Technology 15:815-825 (2001)). The remaining exudate consists largely of sorgoleone congeners differing in the length or degree of saturation of the aliphatic side chain, and in the substitution pattern of the quinone ring (Kagan, et al., J. Agric. Food Chem. 51:7589-7595 (2003); Rimando, et al., J. Nat. Prod. 66:42-45 (2003)). Sorgoleone acts as a potent broad-spectrum inhibitor active against many agronomically important monocot and dicot weed species, exhibits a long half-life in soil, and appears to affect multiple targets in vivo (e.g., Netzly and Butler, 1986; Einhellig and Souza, J. Chemical Ecology 18:1-11 (1992); Nimbal, et al., J. Agric. Food Chem. 44:1343-1347 (1996); Czarnota, et al., 2001; Bertin, et al., 2003; Duke, Trends Biotechnol. 21:192-195 (2003)). Sorgoleone is a promising natural product alternative to synthetic herbicides (Duke, 2003).
The herbicidal and allelopathic properties of sorgoleone make the isolation and characterization of the corresponding genes involved in sorgoleone biosynthesis highly desirable, as manipulation of the pathway in sorghum, or genetic modification of other plant species using these genes could provide important insights into the underlying allelochemical interactions involved (Duke, 2003). Sorgoleone biosynthesis is likely restricted to root hairs, which appear as cytoplasmically dense cells in sorghum, containing large osmiophilic globules deposited between the plasmalemma and cell wall, presumably associated with sorgoleone rhizosecretion (Czarnota, et al., Int. J. Plant Sci. 164:861-866 (2003b); Czarnota, et al., J. Chem. Ecology 29:2073-2083 (2003a)). The biosynthetic pathway of sorgoleone appears to require four types of enzymes: fatty acid desaturases, polyketide synthases, O-methyltransferases, and cytochrome P450 monooxygenases (FIG. 1). Recently, the following enzymes in this pathway have been characterized: two S. bicolor fatty acid desaturases (DES2, DES3; Pan, et al., J. Biol. Chem. 282:4326-4335 (2007) and U.S. Pat. No. 8,383,890), two alkylresorcinol synthases (ARS1, ARS2; U.S. Patent Pub. 2011-0225676), and a 5-n-alk(en)ylresorcinol-utilizing O-methyltransferase (OMT3; U.S. Pat. No. 7,732,666) which likely participate in the biosynthesis of sorgoleone. As of yet, the enzyme or enzymes that convert 3-methyl-5-pentadecatrienyl resorcinol to dihydrosorgoleone, a vital step in the biosynthesis of sorgoleone, have not been isolated or characterized.